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1. From growth to stress: RAF-like kinases as integrators of hormonal signals in plants

   https://doi.org/10.1093/jxb/eraf086  

   Park, Hye Lin; Yoon, Gyeong Mee (March 2025, Journal of Experimental Botany)

   McSteen, Paula (Ed.)

   Abstract RAF-like kinases, members of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, are central integrators of external and internal signals in plant stress responses and growth regulation. These kinases mediate signaling through multiple hormone pathways, including abscisic acid-dependent and -independent pathways, ethylene signaling, and rapid auxin responses. Unlike typical MAPKKKs that function through kinase cascades, RAF-like kinases primarily employ direct phosphorylation of downstream targets and dynamic subcellular localization to mediate specific physiological responses. Here, we review the emerging roles of RAF-like kinases in Arabidopsis thaliana, highlighting their integrative functions in hormone signaling, stress responses, and growth control. The complex interplay between different RAF-like kinase subgroups and their diverse cellular targets underscores the intricate regulatory mechanisms plants have evolved to coordinate environmental responses with development. 

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2. Profiling tyrosine kinase substrate recognition using bacterial peptide display and deep sequencing

   https://doi.org/10.64898/2025.12.28.696772  

   Lee, Minhee; Shah, Neel H (December 2025, bioRxiv)

   Tyrosine kinases control a wide range of cell signaling pathways that are central to human physiology, and they are dysregulated in a variety of human diseases, most notably cancers. Our understanding of tyrosine kinase biology hinges upon a clear delineation of their protein substrates. Thus, much effort has been invested into defining the substrate specificities of tyrosine kinases, which is partly driven by recognition of the amino acid sequences surrounding the phospho-acceptor tyrosine residues. Numerous methods have been developed to profile tyrosine kinase sequence recognition, and these approaches have collectively demonstrated that different tyrosine kinases have distinct substrate sequence preferences. Here, we describe one such method that combines bacterial peptide display and deep sequencing to study tyrosine kinase substrate preferences. Our approach enables rapid measurement of relative phosphorylation efficiencies for thousands of peptides simultaneously. The genetically-encoded nature of the peptide libraries used with this method allows for facile and cheap construction of libraries tailored to answer a variety questions. Notably, our approach is compatible with genetic code expansion via Amber codon suppression, which allows for the construction and screening of libraries containing non-canonical amino acids. Importantly, results from this assay correlate strongly with quantitative measurements of enzyme kinetics, they corroborate previously reported tyrosine kinase substrate preferences, and they can reveal new insights into tyrosine kinase substrate specificity. 

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3. Not your Mother’s MAPKs: Apicomplexan MAPK function in daughter cell budding

   https://doi.org/10.1371/journal.ppat.1010849  

   O’Shaughnessy, William J.; Dewangan, Pravin S.; Paiz, E. Ariana; Reese, Michael L. (October 2022, PLOS Pathogens)

   Kafsack, Bjorn F.C. (Ed.)

   Reversible phosphorylation by protein kinases is one of the core mechanisms by which biological signals are propagated and processed. Mitogen-activated protein kinases, or MAPKs, are conserved throughout eukaryotes where they regulate cell cycle, development, and stress response. Here, we review advances in our understanding of the function and biochemistry of MAPK signaling in apicomplexan parasites. As expected for well-conserved signaling modules, MAPKs have been found to have multiple essential roles regulating both Toxoplasma tachyzoite replication and sexual differentiation in Plasmodium . However, apicomplexan MAPK signaling is notable for the lack of the canonical kinase cascade that normally regulates the networks, and therefore must be regulated by a distinct mechanism. We highlight what few regulatory relationships have been established to date, and discuss the challenges to the field in elucidating the complete MAPK signaling networks in these parasites. 

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4. A kinase to cytokine explorer to identify molecular regulators and potential therapeutic opportunities

   https://doi.org/10.7554/eLife.91472.3  

   Chan, Marina; Kang, Yuqi; Osborne, Shannon; Zager, Michael; Gujral, Taranjit S (February 2024, eLife)

   Cytokines and chemokines are secreted proteins that regulate various biological processes, such as inflammation, immune response, and cell differentiation. Therefore, disruption of signaling pathways involving these proteins has been linked to a range of diseases, including cancer. However, targeting individual cytokines, chemokines, or their receptors is challenging due to their regulatory redundancy and incomplete understanding of their signaling networks. To transform these difficult-to-drug targets into a pharmacologically manageable class, we developed a web-based platform called KinCytE. This platform was designed to link the effects of kinase inhibitors, a well-established class of drugs, with cytokine and chemokine release and signaling networks. The resulting KinCytE platform enables users to investigate protein kinases that regulate specific cytokines or chemokines, generate a ranked list of FDA-approved kinase inhibitors that affect cytokine/chemokine activity, and explore and visualize cytokine signaling network thus facilitating drugging this challenging target class. KinCytE is freely accessible viahttps://atlas.fredhutch.org/kincyte. 

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5. Click chemistry–enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling

   https://doi.org/10.1073/pnas.2025265118  

   Bumpus, Timothy W.; Huang, Shiying; Tei, Reika; Baskin, Jeremy M. (November 2021, Proceedings of the National Academy of Sciences)

   Enzymes that produce second messengers are highly regulated. Revealing the mechanisms underlying such regulation is critical to understanding both how cells achieve specific signaling outcomes and return to homeostasis following a particular stimulus. Pooled genome-wide CRISPR screens are powerful unbiased approaches to elucidate regulatory networks, their principal limitation being the choice of phenotype selection. Here, we merge advances in bioorthogonal fluorescent labeling and CRISPR screening technologies to discover regulators of phospholipase D (PLD) signaling, which generates the potent lipid second messenger phosphatidic acid. Our results reveal glycogen synthase kinase 3 as a positive regulator of protein kinase C and PLD signaling. More generally, this work demonstrates how bioorthogonal, activity-based fluorescent tagging can expand the power of CRISPR screening to uncover mechanisms regulating specific enzyme-driven signaling pathways in mammalian cells. 

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